徳島大学 教育・研究者情報データベース(EDB)

1. 青山学院大学.理工学部
2. Academy of Russia

1. 佐藤 高則

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2. (英) Samejima Tatsuya

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3. (英) Watanabe Machiko

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4. (英) Nogi Shin-ichi

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5. (英) Takahashi Yoshimasa

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6. (英) Kaji Hiroyuki

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7. (英) Teplyakov Alexei

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8. (英) Obmolova Galya

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9. (英) Kuranova Inna

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10. (英) Ishii Keisuke

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(英) Molecular Cloning, Expression and Site-Directed Mutagenesis of Inorganic Pyrophosphatase from Thermus thermophilus HB8

(英) The genomic DNA encoding the inorganic pyrophosphatase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ATCC27634), was isolated by colony hybridization with a probe designed as a part of gene amplified by the PCR method, which was derived from the partial amino acid sequence of the enzyme. The DNA was cloned into a plasmid vector, pUC118, after digestion with BamHI. The inserted nucleotide fragment was about 1.8 kbp in length and the nucleotide sequence included a 525 bp open reading frame. The deduced amino acid sequence was completely identical with that of the enzyme determined by automated Edman analysis of peptide fragments isolated from digests obtained with Staphylococcus aureus V8 protease and Achromobacter protease I, and also from products obtained on chemical cleavage with cyanogen bromide and 70% formic acid. The subunit of this enzyme is composed of 174 amino acid residues with a calculated molecular weight of 19,084. Then, the gene was overexpressed in Escherichia coli BL21 (DE3) using a plasmid vector, pET15b, system. The recombinant enzyme was fully active, and exhibited higher thermostability than the E. coli enzyme. Amino acid residues located on the surface of the recombinant enzyme were determined by means of limited proteolysis, and the results revealed that the environment of Lys residues is almost the same as the crystal structure reported previously [Teplyakov, A. et al. (1994) Protein Sci. 3, 1098-1107]. Furthermore, the roles of two tryptophan residues were investigated by site-directed mutagenesis, which indicated that they may be responsible for the structural integrity and thermostability.

(日) 高度好熱性細菌Thermus thermophilusより，無機ピロリン酸加水分解酵素遺伝子のクロ-ニングを行ない，その全一次構造を決定した． また，大腸菌により発現させた組み換え体酵素は，十分な酵素活性および耐熱性を示した．さらに，プロテア-ゼ限定分解による溶液構造の推定や，部位特異的変異法を用いて作製した変異体の解析から，本酵素の耐熱化に寄与する部位について考察を行なった．

1. (英) Amino Acid Sequence
2. (英) Amino Acid Substitution
3. (英) Base Sequence
4. (英) Cloning, Molecular
5. (英) DNA, Bacterial
6. (英) Enzyme Stability
7. (英) Escherichia coli
8. (英) Hydrolysis
9. (英) Inorganic Pyrophosphatase
10. (英) Molecular Sequence Data
11. (英) Mutagenesis, Site-Directed
12. (英) Pyrophosphatases
13. (英) Recombinant Proteins
14. (英) Sequence Homology, Amino Acid
15. (英) Thermus thermophilus
16. (英) Tryptophan

1. (英) Article.DataBankList.DataBank.DataBankName: GENBANK

2. (英) Article.DataBankList.DataBank.AccessionNumberList.AccessionNumber: AB010580

3. (英) Article.DataBankList.DataBank.AccessionNumberList.AccessionNumber: P38576

4. (英) Article.PublicationTypeList.PublicationType: Journal Article

5. (英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't